toxins Article Phylogeny and Toxin Profile of Freshwater Pufferfish (Genus Pao) Collected from 2 Different Regions in Cambodia Hongchen Zhu 1, Akinori Yamada 1 , Yui Goto 2, Linan Horn 3, Laymithuna Ngy 3, Minoru Wada 1, Hiroyuki Doi 4, Jong Soo Lee 5, Tomohiro Takatani 1 and Osamu Arakawa 1,* 1 Graduate School of Fisheries and Environmental Sciences, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki 852-8521, Japan; [email protected] (H.Z.); [email protected] (A.Y.); [email protected] (M.W.); [email protected] (T.T.) 2 Faculty of Fisheries, Nagasaki University. 1-14, Bunkyo-machi, Nagasaki 852-8521, Japan; [email protected] 3 Faculty of Agronomy, University of Kratie, National Road No. 7, Orussey District, Kratie City, Kratie Province, Cambodia; [email protected] (L.H.); [email protected] (L.N.) 4 Nifrel, Osaka Aquarium Kaiyukan. 2-1, Senribanpakukoen, Suita, Osaka 565-0826, Japan; [email protected] 5 College of Marine Science, Gyeongsang National University, 2, Tongyeonghaean-ro, Tongyeong, Kyungnam 53064, Korea; [email protected] * Correspondence: [email protected]; Tel.: +81-95-819-2844 Received: 15 October 2020; Accepted: 28 October 2020; Published: 30 October 2020 Abstract: The species classification of Cambodian freshwater pufferfish is incomplete and confusing, and scientific information on their toxicity and toxin profile is limited. In the present study, to accumulate information on the phylogeny and toxin profile of freshwater pufferfish, and to contribute to food safety in Cambodia, we conducted simultaneous genetic-based phylogenetic and toxin analyses using freshwater pufferfish individuals collected from Phnom Penh and Kratie (designated PNH and KTI, respectively). Phylogenetic analysis of partial sequences of three mitochondrial genes (cytochrome b, 16S rRNA, and cytochrome c oxidase subunit I) determined for each fish revealed that PNH and KTI are different species in the genus Pao (designated Pao sp. A and Pao sp. B, respectively). A partial sequence of the nuclear tributyltin-binding protein type 2 (TBT-bp2) gene differentiated the species at the amino acid level. Instrumental analysis of the toxin profile revealed that both Pao sp. A and Pao sp. B possess saxitoxins (STXs), comprising STX as the main component. In Pao sp. A, the toxin concentration in each tissue was extremely high, far exceeding the regulatory limit for STXs set by the Codex Committee, whereas in Pao sp. B, only the skin contained high toxin concentrations. The difference in the STX accumulation ability between the two species with different TBT-bp2 sequences suggests that TBT-bp2 is involved in STX accumulation in freshwater pufferfish. Keywords: Cambodia; pufferfish; Pao; phylogenetic analysis; tributyltin-binding protein type 2 (TBT-bp2); saxitoxin (STX) Key Contribution: Werevealed a gene-based phylogeny linked to the toxicity profile of the Cambodian freshwater pufferfish, and suggest for the first time that tributyltin-binding protein type 2 is involved in saxitoxin accumulation in freshwater pufferfish. 1. Introduction Pufferfish of the family Tetraodontidae contain tetrodotoxin (TTX) and/or saxitoxins (STXs), but the toxin ratio differs depending on the genus or species. Marine pufferfish of the genus Takifugu inhabiting Toxins 2020, 12, 689; doi:10.3390/toxins12110689 www.mdpi.com/journal/toxins Toxins 2020, 12, 689 2 of 15 the coastal waters of Japan and Dichtomyctere (formerly known as Tetraodon) living in the brackish waters of Thailand have TTX as their main toxin component [1–3], whereas freshwater pufferfish of the genera Pao and Leiodon (both formerly known as Tetraodon), which inhabit Southeast Asian countries, generally have only STXs [4–7]. On the other hand, Sphoeroides pufferfish from Florida, and Arothron and Canthigaster pufferfish from the Philippines, Japanese coastal waters, or the Caribbean Sea have both TTX and STXs [8–12]. The toxin ratio varies depending on the species, but STXs are dominant in many cases. Marine pufferfish of the genus Takifugu may have small amounts of STXs in addition to TTX [13–15]. Many studies examining the toxification mechanism of pufferfish with TTX revealed that pufferfish do not biosynthesize TTX, but accumulate it through the food chain that starts with TTX-producing bacteria [1]. The accumulated TTX is gradually eliminated if supply via toxic food is not continued [16]. We recently conducted various in vivo TTX administration experiments using nontoxic pufferfish artificially raised with nontoxic diets, and found that the internal TTX kinetics in pufferfish are unique: intestine liver skin/ovary, and change as the pufferfish grows and matures [17–22]. ! ! The pufferfish STX- and TTX-binding protein (PSTBP) isolated from the plasma of Takifugu pardalis by Yotsu-Yamashita et al. [23] is thought to be involved in the internal TTX kinetics, i.e., the absorption, transportation, and accumulation of TTX inside the pufferfish body [24]. PSTBP homologous proteins are widely distributed in TTX-bearing toxic pufferfish of the genera Takifugu and Arothron, but have not been detected in common fish or in nontoxic species of pufferfish [25–27]. The accumulation mechanism of STXs in pufferfish, however, remains unclear, but is presumed to be exogenous via the food chain that starts with STX-producing dinoflagellates in marine environments and STX-producing cyanobacteria in freshwater environments [6,9,11,28]. In a recent in vivo TTX/STX administration experiment using nontoxic cultured pufferfish, Gao et al. [29] found that T. pardalis, which naturally contains TTX, selectively accumulates TTX, and Pao suvattii, which naturally contains STXs, selectively accumulates STXs. In other words, the ratio of TTX/STX in pufferfish appears to depend more strongly on the inherent TTX/STX selectivity of the pufferfish than the prevalence of TTX/STX in prey organisms, but the molecular mechanisms underlying the toxin selectivity remain to be elucidated. Our ongoing genome and transcriptome analyses indicate that the three freshwater pufferfish species, Pao abei, P. suvattii, and Leiodon cutcutia, do not have the PSTBP gene (unpublished data), suggesting that PSTBP is not involved in STX accumulation or selectivity of the genus Pao. Several species of freshwater pufferfish inhabit the rivers and lakes of Cambodia. These pufferfish are considered delicious and there have been many cases of poisoning due to the ingestion of pufferfish caught with other generally edible freshwater fish. According to the Ministry of Health in Cambodia, at least seven poisoning incidents occurred due to freshwater pufferfish consumption from 2017 to 2019, with more than 40 people poisoned and 5 deaths. The Codex Committee on Fish and Fishery Products has set the regulatory limit for STXs as 0.8 mg STX diHCl eq/kg edible tissue [30], · but there is no strict food safety standard for the edible use of freshwater pufferfish in Cambodia. This may be partly due to the fact that the species classification of Cambodian indigenous pufferfish is incomplete and quite confusing, and the limited scientific information on the toxicity and toxin profile. In the present study, to accumulate genetic information on the taxonomy of freshwater pufferfish and the TTX/STX distribution profiles in pufferfish of the family Tetraodontidae, and to contribute to food safety in Cambodia, we conducted simultaneous genetic-based phylogenetic and toxin analyses using freshwater pufferfish specimens collected from two different regions in Cambodia. Moreover, to explore candidate molecules other than PSTBP involved in the absorption, transportation, and accumulation of STXs in freshwater pufferfish, a partial sequence of a putative origin of the molecular evolution of PSTBP [31,32], tributyltin-binding protein type 2 (TBT-bp2), and four commonly used genes (cytochrome b [CYTB], 16S rRNA, cytochrome c oxidase subunit I [COI], and rhodopsin) were used as targets in the phylogenetic analysis. ToxinsToxins 2020, 12, x 689 FOR PEER REVIEW 33 of of 15 15 2. ResultsToxins 2020, 12, x FOR PEER REVIEW 3 of 15 2. Results 2. Results 2.1.2.1. Phylogeny Phylogeny 2.1. Phylogeny AA total total of of 16 16 freshwater freshwater pufferfish pufferfish were were sampled sampled from from small small lakes lakes near near Phnom Phnom Penh Penh (PNH) (PNH) and and KratieKratie (KTI) (KTI)A total (Figures (Figures of 16 freshwater 11 andand2 ,2, and pufferfishand Table Table wereA1 A1).). sampled PartialPartial from sequencessequences small lakes ofof the thenear threethree Phnom mitochondrial mitochondrial Penh (PNH) and genes,genes, CYTB,CYTB,Kratie 16S 16S (KTI)rRNA, rRNA, (Figures and and COI, COI, 1 and were were 2, and determined determined Table A1). forPa forrtial each each sequences sample ofand and the phylogenetically three mitochondrial analyzed analyzed genes, for for taxonomictaxonomicCYTB, identification. identification.16S rRNA, and Phylogenetic COI, Phylogenetic were determined analysis analysis offor 870-bp of each 870-bp sample CYTB CYTB sequencesand sequencesphylogenetically showed showed that analyzed thatthe PNH the for PNH and taxonomic identification. Phylogenetic analysis of 870-bp CYTB sequences showed that the PNH and KTIand sequences KTI sequences each formed each formed a tight cluster a tight together cluster togetherwith Pao withcochinchinensisPao cochinchinensis (AB741980.1)(AB741980.1) or Pao baileyi or KTI sequences each formed a tight cluster together with Pao cochinchinensis (AB741980.1) or Pao baileyi (AB741978.1)Pao baileyi (AB741978.1) (Figure 3). Similar (Figure 3results). Similar were results